• Title/Summary/Keyword: Mixed Disulfide

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Reductive Depolymerization of Bovine Thyroglobulin Multimers via Enzymatic Reduction of Protein Disulfide and Glutathiony­lated Mixed Disulfide Linkages

  • Liu Xi-Wen;Sok Dai-Eun
    • Archives of Pharmacal Research
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    • v.28 no.9
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    • pp.1065-1072
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    • 2005
  • The nascent thyroglobulin (Tg) multimer molecule, which is generated during the initial fate of Tg in ER, undergoes the rapid reductive depolymerization. In an attempt to determine the depolymerization process, various types of Tg multimers, which were generated from deoxy­cholate-treated/reduced Tg, partially unfolded Tg or partially unfolded/reduced Tg, were subjected to various GSH (reduced glutathione) reducing systems using protein disulfide isomerase (PDI), glutathione reductase (GR), glutaredoxin or thioredoxin reductase. The Tg multimers generated from deoxycholate-treated/reduced Tg were depolymerized readily by the PDI/GSH system, which is consistent with the reductase activity of PDI. The PDI/GSH-induced depolymerization of the Tg multimers, which were generated from either partially unfolded Tg or partially unfolded/reduced Tg, required the simultaneous inclusion of glutathione reductase, which is capable of reducing glutathionylated mixed disulfide (PSSG). This suggests that PSSG was generated during the Tg multimerization stage or its depolymerization stage. In particular, the thioredoxin/thioredoxin reductase system or glutaredoxin system was also effective in depolymerizing the Tg multimers generated from the unfolded Tg. Overall, under the net GSH condition, the depolymerization of Tg multimers might be mediated by PDI, which is assisted by other reductive enzymes, and the mechanism for depolymerizing the Tg multimers differs according to the type of Tg multimer containing different degrees and types of disulfide linkages.

Metabolism and Pharmacokinetics of S-(N,N-Diethyldithiocar-bamoyul)-N-acetyl-L-cysteine in Rats

  • Lee, Byung-Hoon;Song, Yun-Seon;Park, Jongsei;Ryu, Jae-Chun
    • Archives of Pharmacal Research
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    • v.17 no.6
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    • pp.428-433
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    • 1994
  • The methabolism and phamacokinetics of a mixed disulfide S-(N, N-diethyldithiocarbamoyl)-N-acetyl-L-cysteine (AC-DDTC) were studied in rats. Two metabolites of AC-DDTC following iv and po administration were indentified in plasma and liver by HPLC and GC, namely N, N-diethyldithiocarbamate (DDTC) and the methyl ester of DDTC (Me-DDTC). AC-DDTC was very unstable in vivo and could not be detected neither in plasma nor in urine. Pharmacokinetic parameters of DDTC following intravenous administration of AC-DDTC (20 mg/kg) were calculated. DDTC has a low affinity to rat tissue and the body clearance was $9.0{\pm}3.4mkl/mim/kg$. The mean residence time (MRT) was $11.5{\pm}16.3 min$. After oral administration of 20 mg/kg AC-DDTC, maximal plasma concenttion ($C_{max}$) was $3.8{\pm}0.2 nmol/ml$ and the bioavailability was 7.04%. $C_{max}$ for DDTC at a dose of 120 mg/kg. AC-DDTC was $40.1{\pm}2.2 nmol/ml$. ART was $47.1{\pm}2.8min$.at a dose of 20 mg/kg and $110.5{\pm}6.0 min$ at 120 mg/kg.

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Intramolecular Disulfide Bonds for Biogenesis of Calcium Homeostasis Modulator 1 Ion Channel Are Dispensable for Voltage-Dependent Activation

  • Kwon, Jae Won;Jeon, Young Keul;Kim, Jinsung;Kim, Sang Jeong;Kim, Sung Joon
    • Molecules and Cells
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    • v.44 no.10
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    • pp.758-769
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    • 2021
  • Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (ICALHM1). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as ICALHM1. Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect ICALHM1 for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both ICALHM1 and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane.

Mixed-type Inhibition of Human Hepatic Cytochrome P450 1-Catalyzed Ethoxyresorufin O-deethylation by Volatile Allyl Sulfides

  • Kim, Hyun-Jung;Chun, Hyang-Sook
    • Food Science and Biotechnology
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    • v.14 no.2
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    • pp.297-300
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    • 2005
  • Effects of allyl sulfides on kinetic behavior of cytochrome P450 1 (CYP1)-catalyzed ethoxyresorufin O-deethylase (EROD) activity were studied using microsomes from benzo[a]pyrene-treated human hepatoma cells. Apparent $K_m$ and $V_{max}$ values were calculated as $2.8\;{\mu}M$ and $3.0\;{\mu}mol$ resorufin/min/mg protein based on Lineweaver-Burk plot of microsomal EROD activity, respectively. Diallyl disulfide (DADS) and diallyl trisulfide (DATS) affected $K_m$ and $V_{max}$ values of EROD activity and acted as mixed-type inhibitors for CYP1 isozymes. Apparent Ki values of DADS and DATS were calculated as 1.07 and 0.88 mM, respectively, by re-plotting slopes of Lineweaver-Burk plot and inhibitor concentrations.

Analysis of S-glutathionylated proteins during adipocyte differentiation using eosin-glutathione and glutaredoxin 1

  • Hwang, Sungwon;Iram, Sana;Jin, Juno;Choi, Inho;Kim, Jihoe
    • BMB Reports
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    • v.55 no.3
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    • pp.154-159
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    • 2022
  • Protein S-glutathionylation is a reversible post-translational modification on cysteine residues forming a mixed disulfide with glutathione. S-glutathionylation, not only protects proteins from oxidation but also regulates the functions of proteins involved in various cellular signaling pathways. In this study, we developed a method for the detection of S-glutathionylated proteins (ProSSG) using eosin-glutathione (E-GSH) and mouse glutaredoxin 1 (mGrx1). ProSSG was efficiently and specifically labeled with E-GSH to form ProSSG-E via thiol-disulfide exchange. ProSSG-E was readily luminescent allowing the detection of ProSSG with semi-quantitative determination. In addition, a deglutathionylation enzyme mGrx1 specifically released E-GSH from ProSSG-E, which increased fluorescence allowing a sensitive determination of ProSSG levels. Application of the method to the adipocyte differentiation of 3T3-L1 cells showed specific detection of ProSSG and its increase upon differentiation induction, which was consistent with the result obtained by conventional immunoblot analysis, but with greater specificity and sensitivity.

Volatile Flavor Compounds Derived from Anchovy Engraulis japonicus Sauce Residues through Maillard Reactions (멸치(Engraulis japonicus) 액젓 부산물로부터 마이야르 반응을 통해 유도 된 휘발성 향기성분)

  • Jin Hyeon Kim;Yong-Jun Cha;Daeung Yu
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.56 no.2
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    • pp.174-181
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    • 2023
  • Volatile flavor compounds of optimal Maillard reactions (MR) derived with the addition of precursors (AP), control (without AP) and raw as anchovy Engraulis japonicus sauce residue were identified and comparatively analyzed using solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS). MR was produced by adding 1% (w/w) glucose and mixed amino acids (threonine 0.543%, glutamic acid 0.194%, glycine 0.382%, w/w) to raw (100 g of anchovy sauce residue and 100 mL of distilled water), and heating at 110 ℃ for 2 h. Among 65 flavor components detected, 7 compounds were produced through Maillard reaction to change in content. A total of 7 volatile flavor compounds, including 2-methylbutanal, 3-methylbutanal, dimethyl disulfide, methylpyrazine, dimethyl trisulfide, methional, and 2-furanmethanol, tended to increase in the order of raw, control, and MR, but methylpyrazine was not detected in control. Amounts of 2-methylbutanal, 3-methylbutanal, dimethyl disulfide, methylpyrazine, dimethyl trisulfide, methional, and 2-furanmethanol having positive odors (dark chocolate-, garlic-, hazelnut-, cooked potato-like) were 11.04, 50.15, 3.25, 8.38, 4.60, 9.59, and 3.08 times higher, respectively, in MR than those in raw.

Essential Cysteine Residues of Yeast Thioredoxin 2 for an electron donor to Thioredoxin Peroxidases

  • Lee, Song-Mi;Kim, Kang-Hwa;Choi, Won-Ki
    • BMB Reports
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    • v.34 no.2
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    • pp.139-143
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    • 2001
  • Thioredoxin (Trx) is a redox protein possessing conserved sequence Cys-Gly-Pro-Cys in ail organisms. Trx acts as an electron donor of many proteins including thioredoxin peroxidase (TPx). Yeast Trx 2 has two redox active cysteine residues at positions 31 and 34. To investigate the redox activity of each cysteine, we generated mutants C31S, C34S, and C31S/C34S using site directed mutagenesis and examined the redox activity of Trx variants as an electron donor for yeast TPx enzymes. None of the three Cysmutated Trx proteins was active as a redox protein in the 5', 5'-dithiobis-(2-dinitrobenzoic acid) reduction under the condition of the presence of NADPH and thioredoxin reductase, and in the thioredoxin dependent peroxidase activity of yeast TPx II. C34S enhanced the glutamine synthetase protection activity of yeast TPx I, even though 100 times more protein was needed to exhibit the same activity to WT. The formation of a mixed disulfide intermediate between Trx and TPx II subunits was analyzed by SDS-PAGE. The mixed dieter form of TPx II was found only for C34S. These results suggest that Cys-31 more effectively acts as an electron donor for TPx enzymes.

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Enhancement of Gene Delivery Using Novel Homodimeric Tat Peptide Formed by Disulfide Bond

  • Lee, Soo-Jin;Yoon, Sung-Hwa;Doh, Kyung-Oh
    • Journal of Microbiology and Biotechnology
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    • v.21 no.8
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    • pp.802-807
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    • 2011
  • Cationic liposomes have been actively used as gene delivery vehicle because of their minimal toxicity, but their relatively low efficiency of gene delivery is the major disadvantage of these vectors. Recently, cysteine residue incorporation to HIV-1 Tat peptide increased liposomemediated transfection compared with unmodified Tat peptide. Therefore, we designed a novel modified Tat peptide having a homodimeric (Tat-CTHD, Tat-NTHD) and closed structure (cyclic Tat) simply by using the disulfide bond between cysteines to develop a more efficient and safe nonviral gene delivery system. The mixing of Tat-CTHD and Tat-NTHD with DNA before mixing with lipofectamine increased the transfection efficiency compared with unmodified Tat peptide and lipofectamine only in MCF-7 breast cancer cells and rat vascular smooth muscle cells. However, cyclic Tat did not show any improvement in the transfection efficiency. In the gel retardation assay, Tat-CTHD and Tat-NTHD showed more strong binding with DNA than unmodified Tat and cyclic Tat peptide. This enhancement was only shown when Tat-CTHD and Tat-NTHD were mixed with DNA before mixing with lipofectamine. The effects of Tat- CTHD and Tat-NTHD were also valid in the experiment using DOTAP and DMRIE instead of lipofectamine. We could not find any significant cytotoxicity in the working concentration and more usage of these peptides. In conclusion, we have designed a novel transfection-enhancing peptide by easy homodimerization of Tat peptide, and the simple mix of these novel peptides with DNA increased the gene transfer of cationic lipids more efficiently with no additional cytotoxicity.

Occupational Neurotoxic Diseases in Taiwan

  • Liu, Chi-Hung;Huang, Chu-Yun;Huang, Chin-Chang
    • Safety and Health at Work
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    • v.3 no.4
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    • pp.257-267
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    • 2012
  • Occupational neurotoxic diseases have become increasingly common in Taiwan due to industrialization. Over the past 40 years, Taiwan has transformed from an agricultural society to an industrial society. The most common neurotoxic diseases also changed from organophosphate poisoning to heavy metal intoxication, and then to organic solvent and semiconductor agent poisoning. The nervous system is particularly vulnerable to toxic agents because of its high metabolic rate. Neurological manifestations may be transient or permanent, and may range from cognitive dysfunction, cerebellar ataxia, Parkinsonism, sensorimotor neuropathy and autonomic dysfunction to neuromuscular junction disorders. This study attempts to provide a review of the major outbreaks of occupational neurotoxins from 1968 to 2012. A total of 16 occupational neurotoxins, including organophosphates, toxic gases, heavy metals, organic solvents, and other toxic chemicals, were reviewed. Peer-reviewed articles related to the electrophysiology, neuroimaging, treatment and long-term follow up of these neurotoxic diseases were also obtained. The heavy metals involved consisted of lead, manganese, organic tin, mercury, arsenic, and thallium. The organic solvents included n-hexane, toluene, mixed solvents and carbon disulfide. Toxic gases such as carbon monoxide, and hydrogen sulfide were also included, along with toxic chemicals including polychlorinated biphenyls, tetramethylammonium hydroxide, organophosphates, and dimethylamine borane. In addition we attempted to correlate these events to the timeline of industrial development in Taiwan. By researching this topic, the hope is that it may help other developing countries to improve industrial hygiene and promote occupational safety and health care during the process of industrialization.